This invention relates to monoclonal antibodies which are reactive with fibrin(ogen) egradation products (FDPs) generated by proteolysis of fibrinogen and cross-linked fibrin with matrix metalloproteinases (MMPs). More particularly, the invention relates to monoclonal antibodies which are reactive with FDPs generated by proteolysis of human fibrin(ogen) with metalloproteinases, including stromelysin, matrilysin, and membrane-type metalloproteinases, such as MMP-3, MMP-7, and MT1-MMP.
Matrix metalloproteinases (MMPs) have the capacity to degrade a number of proteins and proteoglycans that constitute the extracellular matrix of connective tissue. These enzymes participate in the remodeling of tissues in physiological processes such as morphogenesis, embryonic development and angiogenesis, and in the pathophysiology of wound healing, inflammation, tumor invasion, stroke, myocardial infarction, atherosclerosis, arthritis and angiogenesis. The presence of fibrin(ogen)-related antigen (FRA) in vascular and extravascular space has-been described in all of these disease states.
The matrix metalloproteinases (xe2x80x9cMMPsxe2x80x9d or xe2x80x9cmatrixinsxe2x80x9d) are a class of enzymes that are expressed within the connective tissues of vertebrates. The MMPs occur natively in such tissues and play critical roles in the continuous processes associated with the laying down and remodeling of the extracellular matrix (ECM), hence their name: xe2x80x9cmatrixxe2x80x9d metalloproteinases. The MMPs can be characterized as xe2x80x9cintrinsicxe2x80x9d or xe2x80x9cendogenousxe2x80x9d enzymes insofar as their proper function is within the tissues of the organism in which they are natively expressed. As such, the MMPs are distinguished functionally and evolutionarily from the metalloproteinases found in snake venom and the like, which function outside the organism in which they are expressed, and may therefore be designated xe2x80x9cexogenousxe2x80x9d enzymes.
MMPs are known to proteolytically cleave and degrade a number of proteins and proteoglycans that are associated with the extracellular matrix (ECM) of connective tissue. They have been shown to break down a number of proteins including collagen (Types I-IV, VII and X), laminin, fibronectin, elastin and proteoglycans. MMPs have also been identified in leukocytes. It has been shown that MMP-2 and MMP-9 possess elastase activity, to which some of the complex proteolytic activity, initially observed in granulocytes, could be attributed. In addition, MMP-12 has also been shown to possess elastin activity. MMP-7 has been shown to possess strong proteolytic activity and digests the connective tissue protein, aggrecan, cartilage link protein, fibronectin, and elastin. MMPs participate in the remodeling of tissues in physiological processes such as morphogenesis, embryonic development, wound healing and angiogenesis. MMPs are also key actors in the pathophysiology of tumor invasion, joint injuries and joint diseases (synovitides), such as rheumatoid arthritis and osteoarthritis, as well as in gout, inflammatory bowel disease (IBD), pulmonary fibrosis and in stroke and atherosclerotic lesions.
The expression of MMPs and their inhibitors is under extensive and precise cellular control by a variety of molecular mechanisms. Known regulating factors include hormones, cytokines, proto-oncogenes, steroids, and growth factors. MMPs are blocked by specific inhibitors called xe2x80x9ctissue inhibitors of metalloproteinasesxe2x80x9d (TIMPs) that can block the activity of each member of the family. The main focus of research on ECM has been to limit ECM degradation by MMPs to interrupt or interfere with the progression of disease states. Several groups of investigators are making small molecules that could inhibit proteinases to alter their destructive activity in arthritis, and as antiangiogenic factors to inhibit tumor spread.
Matrix metalloproteinase 3 (MMP-3 or stromelysin-1) belongs to the stromelysin class of matrix metalloproteinases. MMP-3 is expressed in mature macrophages, but also in endothelial cells, smooth muscle cells and fibroblasts. More recently, MMP-3 has been shown to be expressed in macrophage-derived foam cells from experimental atheroma. The inactive zymogen, proMMP-3, is activated by neutrophil elastase, plasma kallikrein, plasmin, chymotrypsin, trypsin, cathepsin G, and mast cell tryptase, as well as by mercurial compounds, such as 4-aminophenylmercuric acetate (APMA). Elevated levels of MMP-3 have been found in the joints of patients suffering from osteoarthritis and rheumatoid arthritis. In atherosclerotic plaques there is a large amount of fibrin(ogen)-related antigen (FRA) consisting of different molecular forms. Recent studies have shown the presence of matrix metalloproteinases 3 in atherosclerotic plaques. The known substrates of MMP-3 include proteoglycans, collagen type IV, fibronectin, and laminin.
Membrane-type 1 matrix metalloproteinase (MT1-MMP) belongs to the membrane-type matrix metalloproteinases class of MMP. The membrane-type matrix metalloproteinases are a subclass of the matrix metalloprtoteinase family which uniquely possess a C-terminal transmembrane domain and are initiators of an activation cascade for progelatinase A. Recent studies have shown that they can also efficiently directly degrade a number of matrix macromolecules. Specifically, the expression of MT1-MMP on the cell surface may lead to both progelatinase A activation and extracellular matrix degration. MT1-MMP is expressed in various tissues. MT1-MMP MRNA expression is predominantly expressed in lungs, kidneys, and placenta where extracellular matrix remodeling is relatively active, and lowest in the brain. MT1-MMP is expressed at a very high level in ossifying tissues during embryogenesis where gelatinase A is co-expressed. MT1-MMP expression is elevated in various tumor tissues including lung, gastric, colon, and breast cancers, in which activated gelatinase A is expressed. MT1-MMP also exhibits broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases.
Bini et al, Biochemistry 35 (40): 13056-13063, 1996 showed that both fibrin(ogen) (Fg) and Factor XllIa cross-linked fibrin (XL-Fb) can be substrates for MMPs with some differences among the various classes. MMP-1 (or Collagenase) seems to have little effect on both Fg and XL-Fb. MMP-2 (or Gelatinase A), degrades fibrin(ogen) rapidly and extensively. However, Fg degraded with MMP-2 still retain the ability to form a fibrin clot. On the contrary, Fg degraded with MMP-3 (or Stromelysin-1) was unclottable, as was Fg previously degraded with plasmin. One of the three MMPs, namely MMP-3, was the only physiological enzyme, in addition to plasmin, capable of solubilizing XL-Fb.
To the present time, no stable, sensitive and precise means has existed for detecting fibrin(ogen) degradation products (FDPs) generated by proteolysis of fibrin(ogen) and fibrin with matrix metalloproteinases (MMPs). There has been no suggestion, however, that FDPs generated by MMPs, such as MMP-3, MMP-7, and MT1-MMP proteolysis of fibrinogen and cross-linked fibrin (and not plasmin or other proteases) could be detected. Nor has there been any indication that the detection of FDPs generated by MMPs could be used for determining information associated with rheumatoid arthritis, osteoarthritis, synovitides, angiogenesis, atherosclerosis, renal diseases, inflammation, and malignancy, as well as information associated with fibrin formation and degradation in physiological processes such as in wound healing deficiencies and abnormalities, and in development of human placenta, placenta development, and pregnancy diseases.
As a result, there exists a need for highly specific, sensitive and reproducible monoclonal antibodies that are reactive with FDPs generated by MMPs proteolysis of fibrinogen and cross-linked fibrin (and not plasmin or other proteases). In addition, means for diagnostic testing of subjects with respect to the amount and distribution of FDPs generated by MMPs are needed. The present invention effectively addresses these and other needs for the first time.
The present invention provides monospecific antibodies which specifically bind to fibrin(ogen) degradation products generated by matrix metalloproteinases (MMPs). Particularly preferred MMPs are MMP-3, MMP-7 and MT1-MMP.
The invention further provides a method of detecting a fibrin(ogen) degradation product (FDP) generated by a matrix metalloproteinase (MMP) in a sample, the method comprising: contacting the sample with a monospecific antibody which binds with the fibrin(ogen) degradation product (FDP) generated by the matrix metalloproteinase (MMP), and determining whether there is specific binding of the monospecific antibody in the sample.
The invention yet further provides a kit for the detection of a fibrin(ogen) digestion product (FDP) generated by a matrix metalloproteinase (MMP), comprising: a composition comprising a monospecific antibody which binds with the fibrin) degradation product (FDP) generated by the matrix metalloproteinase (MMP); and a component selected from the group consisting of: a suitable buffer, an FDP standard and a component for detection of the monospecific antibody.
Also provided is a method for diagnosing the presence or probability of a condition, disease or disorder associated with the generation of a fibrin(ogen) digestion product (FDPs) by a matrix metalloproteinase (MMP) in a subject, comprising: providing a sample from the subject, contacting the sample with a monospecific antibody which binds with the fibrin(ogen) degradation product (FDP) generated by the matrix metalloproteinase (MMP), assessing or measuring the specific binding of the monospecific antibody with the sample, and thereby determining the presence or probability of a condition, disease or disorder associated with the generation of the fibrin(ogen) digestion product (FDP) by the matrix metalloproteinase (MMP) in the subject.
Also yet further provided is a method for monitoring a condition, disorder or disease associated with the generation of a fibrin(ogen) digestion product (FDP) by a matrix metalloproteinase (MMP) in a subject, comprising: providing a first sample from the subject to be monitored, contacting the first sample with a monospecific antibody which binds with the fibrin(ogen) degradation product (FDP) generated by the matrix metalloproteinase (MMP), assessing or measuring the specific binding of the monospecific antibody with the first sample from the subject, providing a subsequent sample from the subject, contacting the subsequent sample with the monospecific antibody which binds with the fibrin(ogen) degradation product (FDP) generated by the matrix metalloproteinase (MMP), assessing or measuring the specific binding of the monospecific antibody with the subsequent sample from the subject, and thereby monitoring the condition, disorder or disease associated with the generation of a fibrin(ogen) digestion product (FDP) by a matrix metalloproteinase (MMP) in the subject.